A simple assay for the ribonuclease activity of ribonucleases in the presence of ethidium bromide

The ribonuclease (RNase) activity of ribonucleases has been assayed by observing the change in fluorescence intensity of ethidium bromide on binding with yeast RNA. The binding of EtBr with RNA was monitored via UV-vis and fluorimetric methods. The degradation of RNA by RNase A was monitored by the change in fluorescence emission intensity of ethidium bromide at 600 nm on excitation at 510 nm. The ribonucleolytic activity of RNase A and angiogenin at various pH values was determined by this method. From this technique we have also determined the macroscopic pK_a values of active site residues of these enzymes. This assay permits the evaluation of the catalytic efficiency of enzymatic proteins ranging from high ribonucleolytic activity to low ribonucleolytic activity toward the natural substrate RNA.